2.1. Recombinant Protein Production
Recombinant DNA technology involves insertion of specific DNA sequences into a DNA vehicle called a vector to form a recombinant DNA molecule which is capable of replication in a host cell. Generally, the inserted DNA sequence is foreign to the recipient DNA vehicle, i.e., the inserted DNA sequence and the DNA vector are derived from organisms which do not exchange genetic information in nature, or the inserted DNA sequence may be wholly or partially synthetically made.
Regardless of the method used for construction, the recombinant DNA molecule must be compatible with the host cell, i.e. capable of autonomous replication in the host cell or stably integrated into one or more of the host cell's chromosomes. The recombinant DNA molecule should preferably also have a marker function which allows the selection of the desired recombinant DNA molecule(s) in host cells. In addition, if all of the proper replication, transcription, and translation signals are correctly arranged on the recombinant vector, the foreign gene will be properly expressed in, e.g. the transformed bacterial cells, or in permissive cell lines or hosts infected with a recombinant virus or carrying a recombinant plasmid having an appropriate origin of replication.
Different genetic signals and processing events control levels of gene expression such as DNA transcription and messenger RNA (mRNA) translation. Transcription of DNA is dependent upon the presence of a promoter, which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eukaryotic promoters differ from those of procaryotic promoters. Furthermore, eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in procaryotic systems and furthermore, procaryotic promoters are not recognized and do not function in eucaryotic cells.
Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals, which differ from those of eukaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno (S/D) sequence on the mRNA [Shine and Dalgarno, Nature 254:34 (1975)]. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually ATG, which encodes the amino-terminal methionine of the protein. The S/D sequences are complementary to the 3' end of the 16S ribosomal RNA (rRNA), and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome on the mRNA.
Although the Shine/Dalgarno sequence, consisting of the few nucleotides of complementarity between the 16S ribosomal RNA and mRNA, has been identified as an important feature of the ribosome binding site [Shine and Dalgarno, id.; Steitz in Ribosomes: Structure, Function and Genetics, ed. Chambliss et al., Baltimore, Md., University Park Press, pp. 479-495 (1980)], computer analysis has indicated that approximately one hundred nucleotides surrounding the ATG initiating codon are involved in ribosome/mRNA interaction as indicated by proper prediction of translation start signals [Stormer et al., Nucl. Acids Res. 10: 2971 (1982); Gold et al., Proc. Natl. Acad. Sci. 81: 7061 (1984)].
Successful expression of a cloned gene thus requires sufficient transcription of DNA, translation of the mRNA and possibly post-translational modification of the protein using the host cell synthetic machinery for both mRNA synthesis and protein synthesis.